Pathogenicity in Verticillium on strawberry plants

In the most common strawberry cv. ’Elsanta’, Verticillium infection can lead to rapid wilt and even death of plants. It is known, that a dead plant can be located directly beside vital ones. In a survey of 8 fields in Brandenburg, Mecklenburg- Vorpommern, Sachsen-Anhalt and Sachsen, 432 genotypes of Verticillium dahliae Kleb. were isolated from wilted and even vital plants from 8 fields and classified by PCR-fingerprints. For strawberries, the genotypes can be classified as apathogenic, weakly and highly pathogenic according to the results of climate chamber experiments on strawberry transplants. At landscape scale, similarity analysis of the PCR fingerprints of 432 genotypes resulted in 13 genetic subtypes. Several of these subtypes occurred at all fields, whereas 1 subtype was found in one location only. At field scale, 2 to 11 different subtypes per field were observed. Vital plants were colonised by up to 9 subtypes, wilted plants by up to 11 subtypes. Population structure of Verticillium subtypes is different between vital and wilted plants, the same subtypes can occur in either plant group. In our plot experiments, wilt symptoms could be reduced by changing the Verticillium population structure in the plant. Inoculation of plants with a mixture of three Verticillium genotypes sustained plant vitality over a period of 15 months (WO 2007/051654)

Evaluation of the Antimicrobial Activity of Endophytic Bacterial Populations From Chinese Traditional Medicinal Plant Licorice and Characterization of the Bioactive Secondary Metabolites Produced by Bacillus atrophaeus Against Verticillium dahliae

Endophytic bacteria associated with medicinal plants possess unique strategies that enhance growth and suvival of host plants, many of which are mediated by distinctive secondary metabolites. These bacteria and their secondary metabolites are important subjects for both basic and applied research aimed at sustainable agriculture. In the present study, 114 endophytic strains isolated from the wild ethnomedicinal plant Glycyrrhiza uralensis (licorice) were screened for their in vitro antimicrobial activities against common fungal pathogens of tomato (Fusarium oxysporum f. sp., Fulvia fulva, Alternaria solani), cotton (Fusarium oxysporum f. sp. Vesinfectum, Verticillium dahliae), pomegranite (Ceratocystis fimbriata), Cymbidinium (Colletotrichum gloeosporioides), and Tsao-ko (Pestalotiopsis microspora and Fusarium graminearum) and the common bacteria Staphylococcus aureus, Bacillus cereus, Salmonella enteritidis, and Escherichia coli. Several Bacillus strains, particularly Bacillus atrophaeus and Bacillus mojavensis, had a broad spectrum of antifungal and antibacterial activity. A total of 16 strains, selected based on broad antimicrobial activity, were shown to contain at least one putative secondary metabolite-encoding gene (i.e., polyketide synthase or non-ribosomal peptide synthetase) and/or one lytic enzyme (i.e., protease, cellulase, lipase, chitinase), which may be important mediators of antagonistic activity against pathogens. Five strains, representing Bacillus atrophaeus and Bacillus mojavensis, were selected for plant growth chamber experiments based on strong in vitro antifungal activities. All five strains significantly reduced disease severity in Arabidopsis thaliana plants challenged with V. dahlia infection. Gas-chromatography/mass-spectrometry analysis of cell-free extracts of Bacillus atrophaeus strain XEGI50 showed that at least 13 compounds were produced only during co-cultivation with V. dahlia, including putative compounds known to have antimicrobial activity, such as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester; 9,12-octadecadienoic acid (Z,Z)-, methyl ester; 9-octadecenoic acid, methyl ester, (E)-; and decanedioic acid, bis(2-ethylhexyl) ester. To our knowledge, this study is the first to report that bacteria isolated from G. uralensis have biocontrol abilities. Our findings provide new insights into the antimicrobial activities of natural endophytes, particularly B. atrophaeus, and suggest this species may a promising candidate as a biocontrol agent to confer resistance to Verticillium wilt disease and other phytopathogens in cotton and other crops

Single-molecule real-time sequencing combined with optical mapping yields completely finished fungal genome

Next-generation sequencing (NGS) technologies have increased the scalability, speed, and resolution of genomic sequencing and, thus, have revolutionized genomic studies. However, eukaryotic genome sequencing initiatives typically yield considerably fragmented genome assemblies. Here, we assessed various state-of-the-art sequencing and assembly strategies in order to produce a contiguous and complete eukaryotic genome assembly, focusing on the filamentous fungus Verticillium dahliae. Compared with Illumina-based assemblies of the V. dahliae genome, hybrid assemblies that also include PacBio- generated long reads establish superior contiguity. Intriguingly, provided that sufficient sequence depth is reached, assemblies solely based on PacBio reads outperform hybrid assemblies and even result in fully assembled chromosomes. Furthermore, the addition of optical map data allowed us to produce a gapless and complete V. dahliae genome assembly of the expected eight chromosomes from telomere to telomere. Consequently, we can now study genomic regions that were previously not assembled or poorly assembled, including regions that are populated by repetitive sequences, such as transposons, allowing us to fully appreciate an organism’s biological complexity. Our data show that a combination of PacBio-generated long reads and optical mapping can be used to generate complete and gapless assemblies of fungal genomes. IMPORTANCE Studying whole-genome sequences has become an important aspect of biological research. The advent of nextgeneration sequencing (NGS) technologies has nowadays brought genomic science within reach of most research laboratories, including those that study nonmodel organisms. However, most genome sequencing initiatives typically yield (highly) fragmented genome assemblies. Nevertheless, considerable relevant information related to genome structure and evolution is likely hidden in those nonassembled regions. Here, we investigated a diverse set of strategies to obtain gapless genome assemblies, using the genome of a typical ascomycete fungus as the template. Eventually, we were able to show that a combination of PacBiogenerated long reads and optical mapping yields a gapless telomere-to-telomere genome assembly, allowing in-depth genome sanalyses to facilitate functional studies into an organism’s biology

Complete mitochondrial genome of the Verticillium-wilt causing plant pathogen Verticillium nonalfalfae

Verticillium nonalfalfae is a fungal plant pathogen that causes wilt disease by colonizing the vascular tissues of host plants. The disease induced by hop isolates of V. nonalfalfae manifests in two different forms, ranging from mild symptoms to complete plant dieback, caused by mild and lethal pathotypes, respectively. Pathogenicity variations between the causal strains have been attributed to differences in genomic sequences and perhaps also to differences in their mitochondrial genomes. We used data from our recent Illumina NGS-based project of genome sequencing V. nonalfalfae to study the mitochondrial genomes of its different strains. The aim of the research was to prepare a V. nonalfalfae reference mitochondrial genome and to determine its phylogenetic placement in the fungal kingdom. The resulting 26,139 bp circular DNA molecule contains a full complement of the 14 "standard" fungal mitochondrial protein-coding genes of the electron transport chain and ATP synthase subunits, together with a small rRNA subunit, a large rRNA subunit, which contains ribosomal protein S3 encoded within a type IA-intron and 26 tRNAs. Phylogenetic analysis of this mitochondrial genome placed it in the Verticillium spp. lineage in the Glomerellales group, which is also supported by previous phylogenetic studies based on nuclear markers. The clustering with the closely related Verticillium dahliae mitochondrial genome showed a very conserved synteny and a high sequence similarity. Two distinguishing mitochondrial genome features were also found-a potential long non-coding RNA (orf414) contained only in the Verticillium spp. of the fungal kingdom, and a specific fragment length polymorphism observed only in V. dahliae and V. nubilum of all the Verticillium spp., thus showing potential as a species specific biomarker

Results from a three year testing project of new strawberry cultivars in Verticillium infested soils and under organic farming conditions

As part of a research project 13 cultivars were planted in 2005 at 11 sites on 9 farms in 5 different Austrian regions. The aim was to find new cultivars tolerant to soil-borne pathogens and leaf/fruit diseases, with high yield, winter hardness and good fruit quality, to serve as alternative to the highly susceptible cultivar ‘Elsanta’, regarding soil-borne diseases. Plant vigour and infestation with Verticillium dahliae and leaf diseases were evaluated in 2005 und 2006 at 7 sites and in 2007 at 3 sites. In addition, following parameters were assessed on 2 organically managed sites in 2006 and 2007: marketable yield, percentage of different categories of unmarketable fruits and incidence of the blossom weevil. In 2006 fruit characteristics and consumer acceptance were studied. ‘Elsanta’ showed the highest infestation with V. dahliae whereas ‘Salsa’, ‘Daroyal’ and ‘Alice’ were most tolerant. ‘Dora’, ‘Eva’, ‘Queen Elisa’ and ‘Daroyal’ recorded significantly higher losses by the blossom weevil than ‘Alice’. ‘Alba’ and ‘Divine’ were the earliest cultivars in ripening time. Highest marketable yield per plant had the late ripening cultivars, particularly ‘Salsa’ and ‘Sonata’. Of all early ripening cultivars tested, ‘Elsanta’ showed the highest productivity, followed by 'Alba', ‘Darselect’, ‘Daroyal’ and ‘Eva’. Regarding fruit firmness, content of ascorbic acid, shelf life and appearance, ‘Alba’, ’Clery’, ‘Eva’ and ‘Queen Elisa’ were most convincing. The best tasting cultivars were ‘Clery’, ‘Daroyal’ and ‘Divine’. Summing up all the results, ‘Alba’, ‘Alice’ and ‘Salsa’ can be recommended for organic production, whereas ‘Clery’, ‘Daroyal’, ‘Darselect’, ‘Elsanta’, ‘Eva’, ‘Queen Elisa’ and ‘Sonata’ are classified as suitable for only a limited extent. ‘Elsanta’, ‘Divine’, ‘Dora’ and ‘Sonata’ are particularly unsuitable for growing in Verticillium infested soils. ‘Divine’, ‘Dora’, and ‘Record’ cannot be recommended for organic production at all

First record of Verticillium dahliae on olive in Malta

Disease note describing the first record of Verticillium dahliae on olive in Maltapeer-reviewe

Low temperature/short duration steaming as a sustainable method of soil disinfection

This report was presented at the UK Organic Research 2002 Conference. Soil samples containing resting structures of fungal crop pathogens (Verticillium dahliae, Sclerotinia sclerotiorum, Sclerotium cepivorum, Pythium ultimum), potato cyst nematodes (Globodera rostochiensis and Globodera pallida) and weeds (Chenopodium album and Agropyron repens) were treated with aerated steam in the laboratory at temperatures ranging from 50–80oC in a specially constructed apparatus. Steaming at 50 or 60oC for three minutes, followed by an eight-minute resting period in the steamed soil and immediate removal from the soil thereafter, resulted in 100% kill of all weeds, fungi and nematodes. Low temperature/ short duration soil steaming could become a sustainable alternative to chemical or high-temperature steam soil disinfestation

Perspectives of Anaerobic Soil Disinfestation

Biological soil disinfestation is an environmentally friendly method to disinfest soil. From now on we refer to it as anaerobic soil disinfestation (ASD). With ASD a green manure crop (40 t/ha) is homogeneously incorporated into the topsoil (0-30 cm) after which the field is lightly compacted and irrigated. Subsequently the field is mechanically covered with air tight virtually impermeable film (VIF) to restrict oxygen supply to the topsoil. The soil is left covered for six weeks in the summer. In this period anaerobic conditions develop rapidly in which toxic fermentation products are formed. These conditions are responsible for elimination of fungal and bacterial pathogens as well as parasitic nematodes, insects and weeds. In the Netherlands since 2004 anaerobic soil disinfestation (ASD) was applied on approximately 70 ha mainly for asparagus and strawberry runners production. When the asparagus crop was replanted ASD proved to be profitable for many years by controlling Fusarium oxysporum. Although highly effective against soilborne pathogens and pests ASD is not applied widely in the Netherlands due to the high costs of about € 4000 per ha. Apart from the costs this technique needs special attention to prevent plastic damage. Moreover the mechanism behind ASD is not well defined which makes recommendations for growers complex. A new and promising development in ASD is the application of defined products on a basis of mixtures of carbon hydrates and proteins. The mechanism of ASD will be studied in the next few years with some of these products by measuring gases and fatty acids and microbial shifts in relation to efficacy. Chemical soil disinfestation with fumigants is restricted more and more and a non-chemical approach of soil disinfestation like ASD is urgently neede